Sex-specific death in the Asian corn borer moth(Ostrinia furnacalis) infected with Wolbachiaoccurs across larval development

Hironori Sakamoto, Daisuke Kageyama, Sugihiko Hoshizaki, and Yukio Ishikawa

Abstract: Maternally inherited endosymbiotic bacteria of the genus Wolbachia induce various kinds of reproductive altera-tions in their arthropod hosts. In a Wolbachia-infected strain of the adzuki bean borer moth, Ostrinia scapulalis (Lepidop-tera: Crambidae), males selectively die during larval development, while females selectively die when Wolbachia areeliminated by antibiotic treatment. We found that naturally occurring Wolbachia in the congener O. furnacalis caused sex-specific lethality similar to that in O. scapulalis. Cytogenetic analyses throughout the entire larval development clarifiedthat the death of males (when infected) and females (when cured) took place mainly during early larval stages. However,some individuals also died after complete formation of larval bodies but before egg hatching, or at late larval stages, evenin the penultimate instar. Although the specific timing was highly variable, death of males and females occurred before pu-pation without exception. The potential association of sex-specific lethality with the sex determination mechanism wasalso examined and is discussed.

Key words: feminization, male killing, Ostrinia furnacalis, sex-specific lethality, Wolbachia.

Resume : Des bacteries endosymbiotiques du genre Wolbachia, lesquelles sont transmises maternellement, induisent diffe-rentes alterations reproductives chez les arthropodes qui leur servent d’hotes. Chez une souche infectee de Wolbachia del’Ostrinia scapulalis (Lepidoptera : Crambidae), les males meurent de facon selective au cours du developpement larvairetandis que les femelles meurent selectivement lorsque les Wolbachia sont elimines par un traitement antibiotique. Les au-teurs ont trouve que des Wolbachia presents naturellement chez une espece proche, l’O. furnacalis, cause egalement uneletalite selon le sexe semblable a celle observee chez l’O. scapulalis. Des analyses cytogenetiques au cours de l’ensembledu developpement larvaire a permis de determiner que la mort des males (lorsque infectes) et des femelles (lorsque traites)survenait principalement lors des premiers stades du developpement larvaire. Cependant, certains individus decedaient ega-lement avant l’eclosion, apres avoir complete la formation des corps larvaires ou aux stades tardifs et meme l’avant-dernierstade larvaire. Bien que le moment precis variait grandement, la mort des males et femelles survenait avant la pupaisonsans exception. L’association potentielle de la letalite specifique a un sexe et le mecanisme de determination du sexe a eteexaminee et est discutee.

Mots-cles : feminisation, mortalite male, Ostrinia furnacalis, letalite specifique a un sexe, Wolbachia.

[Traduit par la Redaction]

Introduction

Male killing occurs in a variety of insects and is oftencaused by maternally inherited endosymbionts. Symbiont-induced male killing has been conventionally classifiedinto 2 categories: ‘‘early male killing’’, wherein embryosor young larvae are killed, and ‘‘late male killing’’,

wherein mature larvae are killed (Hurst 1991, 1993; Hurstand Majerus 1993). Early male killing, observed frequentlyin insects such as ladybird beetles, butterflies, moths, andfruit flies, is caused by endosymbiotic bacteria of diversetaxa including Spiroplasma, Rickettsia, Wolbachia, and Ar-senophonus (O’Neill et al. 1997; Hurst and Jiggins 2000;Bourtzis and Miller 2003). In contrast, late male killing,known only in mosquitoes, is caused by unicellular eukar-yotes of the phylum Microsporidia (Hurst 1991; Dunn andSmith 2001).

A group of endosymbiotic bacteria belonging to the genusWolbachia has been found in a wide variety of arthropodsand filarial nematodes. Wolbachia is of particular interestbecause it can induce various kinds of reproductive altera-tions in its hosts, such as early male killing, feminization,induction of parthenogenesis, and cytoplasmic incompatibil-ity (reviewed by Werren 1997; Bourtzis and O’Neill 1998;Stouthamer et al. 1999). In lepidopteran insects, naturallyoccurring Wolbachia can cause feminization (Hiroki et al.2002), early male killing (Hurst et al. 2000; Jiggins et al.2000; Dyson et al. 2002; Mitsuhashi et al. 2004), or cyto-

Received 9 February 2007. Accepted 22 May 2007. Publishedon the NRC Research Press Web site at genome.nrc.ca on20 July 2007.

Corresponding Editor: W. Traut.

H. Sakamoto,1,2 S. Hoshizaki, and Y. Ishikawa. GraduateSchool of Agricultural and Life Sciences, The University ofTokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan.D. Kageyama. National Institute of Agrobiological Sciences,Owashi 1-2, Tsukuba, Ibaraki 305-8634, Japan.

1Corresponding author (e-mail: hirowol@yahoo.co.jp).2Present address: Kannondai 3-1-3, Tsukuba, Ibaraki 305-8604,Japan.

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plasmic incompatibility (Sasaki and Ishikawa 1999) depend-ing on the host–Wolbachia system.

In the adzuki bean borer moth, Ostrinia scapulalis (Lepi-doptera: Crambidae), naturally occurring Wolbachia appa-rently causes male killing (Kageyama and Traut 2004).However, antibiotic treatment of infected larvae, which isexpected to restore a 1:1 sex ratio, results in the generationof all-male progeny as a result of female killing in the sub-sequent generation (Kageyama and Traut 2004). Moreover,when larvae were incompletely cured of Wolbachia, pheno-typically sexually mosaic moths having exclusively malegenotypes (hereafter, referred to simply as ‘‘sexual mo-saics’’) appeared (Kageyama and Traut 2004). Based onthese findings, Kageyama and Traut (2004) hypothesizedthat Wolbachia has the ability to feminize genetic males ofthe host, and that complete feminization of genetic males isfatal. This hypothesis appears to explain the findings well;however, given that Kageyama and Traut (2004) examinedthe genetic sexes only in hatchlings and the last larval stage,the timing of male killing and female killing, which is im-portant for inferring the mechanisms of killing, remainedunknown.

Here we report that naturally occurring Wolbachia in theAsian corn borer moth, Ostrinia furnacalis, a congener ofO. scapulalis, also causes sex-specific lethality. Using cyto-genetic techniques, we discovered that the timing of malekilling (when infected) and female killing (when cured) ismore variable than previously thought, extending across awide range of the larval development. Similarities and dif-ferences with respect to pathological and temporal aspectsof male killing and female killing are discussed in the con-text of developmental and evolutionary biology.

Materials and methods

InsectsFemale O. furnacalis were collected in Akiruno, Tokyo,

Japan, in 2005, and individually housed in plastic cups.After females had laid eggs, the Wolbachia infection waschecked by diagnostic polymerase chain reaction (PCR; seebelow). The offspring of the single wild-caught females (in-fected and uninfected) were reared on a commercial diet(Silkmate-2MTM, Nosan Corp., Yokohama, Japan) at 25 8Cunder a 16 h light and 8 h dark photoperiod. Three Wolba-chia-infected maternal lines (AK5014, AK5035, andAK5062) and 1 uninfected line (AK5061) were maintainedand used for examination. Larvae used for genetic sexingwere reared individually to avoid possible effects of canni-balism. The age of larvae is expressed as the number ofdays since hatching, where the day of hatching is day 0.

Diagnostic PCR for Wolbachia infectionGenomic DNA was purified from the ovaries of adult fe-

males using a QIAamp DNA Mini Kit (QIAGEN, Hilden,Germany). The presence or absence of Wolbachia DNAwas tested by PCR using TaKaRa Ex TaqTM (Takara-BioInc., Ohtsu, Japan) and primers specific for Wolbachia 16SrDNA (99F and 994R from O’Neill et al. 1992). PCR cy-cling conditions were 94 8C for 5 min followed by 30 am-plification cycles of 94 8C for 1 min, 55 8C for 1 min, and72 8C for 1 min and a final extension at 72 8C for 10 min.

The products were separated on 2% agarose gels to checkfor the presence or absence of a DNA band of the expectedsize (895 bp).

Antibiotic treatment of larvaeCured (Wolbachia-eliminated) adult females were ob-

tained by feeding the larvae of the infected lines AK5035and AK5062 a diet containing tetracycline hydrochloride(0.06% w/w) throughout the entire larval stage. The elimina-tion of Wolbachia was confirmed using diagnostic PCR onDNA extracted from adult ovarian tissue.

Antibiotic treatment of adultsWolbachia-infected female moths were allowed to mate

with uninfected males in a screened cage (20 cm �20 cm � 20 cm) for a period of 2 days. To produce sexualmosaics, ion-supplemented water (Pocari Sweat1, OtsukaPharmaceutical, Tokyo, Japan) containing tetracycline hy-drochloride (0.24% w/v) was continuously given to theWolbachia-infected females after the males were removed.Six females proved fertile after the treatment. Eggs werecollected daily, and eggs laid on different days were rearedseparately.

Observation of the sex chromatin for genetic sexingThe presence or absence of sex chromatin in the inter-

phase nuclei allowed us to diagnose the genetic sex, WZ orZZ, at the early developmental stages and in adults. Midgutsand silk glands were isolated from first-instar larvae by pull-ing the abdominal tip with fine forceps. Malpighian tubulesof 10-day-old larvae, 20-day-old larvae, and adults were dis-sected out in sterile saline. These tissues were placed onslides and fixed by treating with methanol : acetic acid(3:1) for approximately 1 min. The preparations werestained with lactic acetic orcein and examined under a lightmicroscope as described by Kageyama and Traut (2004).

Results

Sex chromosome systemSex chromatin was found in all uninfected adult females

examined (N = 30; Fig. 1a), and no sex chromatin wasfound in the uninfected males examined (N = 30; Fig. 1b).These findings indicated that O. furnacalis has a female-heterogametic sex chromosome constitution (WZ in fe-males, ZZ in males), like the majority of lepidopteran spe-cies (reviewed by Traut and Marec 1996) includingO. scapulalis (Kageyama and Traut 2004). Sex chromatinwas found in all examined Wolbachia-infected adult fe-males (N = 30; Fig. 1c), which indicated that the infectedfemales were genetically female and excluded the possibilityof feminization of genetic males into functional females.

Absence of sex-specific mortality in the uninfected lineIn the uninfected line, most eggs (758/763) developed

fully, and completely formed larval bodies could be ob-served through the eggshells under a dissecting microscope.However, 65 eggs failed to hatch. Among the unhatched lar-vae, 26 of the 54 individuals examined possessed sex chro-matin, indicating that the genetic sex ratio was notsignificantly distorted from 1:1 (P > 0.05). Likewise, nearly

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half of the living larvae examined at different ages (154 of300 on day 0, 54 of 112 on day 10, and 70 of 152 on day20) possessed sex chromatin (Table 1). In addition to nor-mally developing larvae, a small number of developmentallyretarded larvae (dwarfs) were observed on day 20 (14 of166). Despite being small, some of these individuals werein the fourth (penultimate) instar, which was confirmed bythe number of head capsule exuviae. The presence of sexchromatin indicated that 4 of the dwarfs were genetically fe-male (WZ; Table 1). These results confirm that the geneticsex ratio (WZ:ZZ) was approximately 1:1 throughout thelarval stage, and sex-specific mortality did not occur in theuninfected line.

Male killing during larval development in the infectedlines

In the infected lines (AK5014, AK5035, and AK5062), al-

most all adults were female (Table 2). Complete or nearlycomplete larval body formation was observed in most eggs(932/941), but 113 failed to hatch. Among the unhatchedlarvae, 26 of 102 individuals examined possessed sex chro-matin, indicating that the genetic sex ratio was significantlybiased toward males (ZZ; P < 0.001). On day 0, the geneticsex ratio of the hatched larvae was not significantly differentfrom 1:1 (215 WZ and 185 ZZ; P > 0.05); however, it wassignificantly biased toward females on day 10 (91 WZ and17 ZZ; P < 0.001). On day 20, all 95 larvae of normal sizewere genetically and phenotypically female, as determinedby the presence of sex chromatin and the absence of testes,respectively (Table 3). Nine dwarf larvae, some of whichwere determined to be in the fourth instar, were observedon day 20. The observation of sex chromatin showed that 3of 5 dwarfs examined were genetically male (ZZ; Table 3).The 4 unexamined dwarfs died by day 28. These results

Fig. 1. Sex chromatin in interphase nuclei of adult Ostrinia furnacalis. Malpighian tubule cells with highly polyploid branched nucleistained with lactic acetic orcein, from an uninfected female (a), an uninfected male (b), and an infected female (c). Magnifications in a–care equal. Bursa copulatrix cells from an uninfected female (d) and an individual with the sexual mosaic phenotype generated by tetracy-cline treatment (e). Arrows indicate sex chromatin bodies.

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suggest that in the infected lines, the timing of male killingvaried from just before hatching to >20 days after hatching,thus spanning almost the entire period of larval development.

Female killing during larval development in the curedlines

Progeny produced by females of Wolbachia-infected lines(AK5035 and AK5062) that had been treated with tetracy-cline throughout the larval stage were exclusively males atthe adult stage (Table 2). In the cured lines, most eggs(696/707) developed a complete larval body. However, 59eggs failed to hatch. Among the unhatched larvae, 47 of 56individuals examined possessed sex chromatin, indicatingthat the genetic sex ratio was significantly biased toward fe-males (WZ; P < 0.001). The genetic sex ratio of the hatchedlarvae was not significantly different from 1:1 (144 WZ and156 ZZ; P > 0.05) on day 0, but shifted to significantlymale-biased (6 WZ and 83 ZZ; P < 0.001) on day 10. Onday 20, all 89 larvae of normal size were genetically andphenotypically male, as determined by the absence of sexchromatin and the presence of testes, respectively (Table 4).In addition to the normal-sized larvae, 4 dwarf larvae wereobserved on day 20. The observation of sex chromatinshowed that these 4 larvae were genetically male (ZZ; Ta-ble 4). These results indicate that in the cured lines, all fe-males died by day 20 but some female larvae survived atleast until day 10.

Appearance of sexual mosaics with male genotypesAmong the progenies produced by tetracycline-treated

mothers, eggs laid on the first and second days of treatmentdeveloped exclusively into female adults (the sum of 6broods is shown in Table 5). Eggs laid on the third andfourth days developed as females, sexual mosaics, or males.The sexual mosaics were readily recognizable from the colorpattern on their wings. Twenty mosaics were dissected, andMalpighian tubules, midgut, and bursa copulatrix were ex-amined for their sex chromatin status. The female-specificbursa copulatrix was found in 10 of 20 mosaics. The sexchromatin was not found in any tissue of any mosaic exam-ined, indicating that the mosaics were all genetically male(ZZ, Fig. 1e; the nuclei of bursa copulatrix of a normal fe-male are shown in Fig. 1d as controls). Of the 20 mosaics, 5had both testes and a bursa copulatrix, 10 had testes only,and 5 had only the bursa copulatrix. No individuals had ova-ries.

Discussion

Our observations demonstrate that in several lines of theAsian corn borer (O. furnacalis) infected with the Wolbachiastrain wFur, (i) complete male killing occurs, (ii) femalekilling occurs when Wolbachia is eliminated, and (iii) sexualmosaics with male genotype are produced from femalesthat are incompletely cured of the Wolbachia infection.These features are essentially identical to those previouslyreported in the adzuki bean borer moth (O. scapulalis) infectedwith the Wolbachia strain wSca (Kageyama and Traut2004). We therefore revise the previous conclusion thatwFur causes feminization of genetic males of O. furnacalis

Table 1. Genetic sex ratios (WZ:ZZ) of larvae produced by 6 uninfected female O. furnacalis (line AK5061).

Proportion of eggs Genetic sex ratio (WZ:ZZ) of larvae

Brood Wolbachia infection Matured Hatched N Unhatched Day 0 Day 10 Day 20a

1 Negative 1.00 0.97 101 2:1 26:24 20:15 n.e.2 Negative 0.98 0.87 135 9:6 32:18* n.e. 16:34* (2:3)3 Negative 0.98 0.95 128 1:1 19:31 21:26 n.e.4 Negative 1.00 0.88 115 3:5 30:20 13:17 5:3 (0:2)5 Negative 1.00 0.90 148 6:8 21:19 n.e. 23:27 (2:4)6 Negative 1.00 0.90 136 5:7 26:24 n.e. 26:18 (0:1)Total 1.00 0.91 763 26:28 154:146 54:58 70:82 (4:10)

Note: Genetic sex ratios were inferred by the presence (WZ, female) or absence (ZZ, male) of sex chromatin. Wolbachia in-fection in each brood was checked by diagnostic PCR (30 samples/brood). N, number of eggs examined; n.e., not examined. Sig-nificant deviations from 1:1 are indicated with * (P < 0.05), as determined by a �2 test.

aIn addition to ratios for normal-sized individuals, those for developmentally retarded individuals are shown separately inparentheses.

Table 2. Sexual phenotypes of the offspring produced by Wolbachia-infected and curedfemales of O. furnacalis.

No. of offspring

Line (no. of females) Wolbachia infection Female Mosaic Male

AK5014 (6) Infected 350 0 5.a

AK5035 (4) Infected 172 0 0.AK5062 (8) Infected 517 0 15.AK5035 (2) Curedb 0 0 119.AK5062 (2) Curedb 0 0 110.

aAll 5 males were Wolbachia-negative by diagnostic PCR.bLarvae were fed a diet containing tetracycline throughout the larval stage.

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(Kageyama et al. 1998, 2002) to state that genetic males ofO. furnacalis are feminized by wFur but die during larvaldevelopment. By analyzing individual genetic sexes duringlarval development, we have provided insight into the tim-ing and the characteristics of sex-specific mortality associ-ated with Wolbachia infection.

Timing of male killingKageyama and Traut (2004) showed that in a Wolbachia-

infected line of O. scapulalis, some males died before egghatching and the remaining males died after egg hatching;

however, the specific timing of death was not examined indetail. We simply assumed that male killing occurs specifi-cally at the stage immediately prior to or shortly after egghatching. To our surprise, however, our analyses of geneticsexes during larval development showed that the timing ofmale killing could vary considerably in O. furnacalis linesinfected with Wolbachia (Table 3). We observed that mostmales died just after hatching or in the eggshells owing tohatching failure. Some larvae underwent molting and sur-vived as late as the fourth larval stage; however, without ex-ception, all died before pupation.

In the butterfly Acraea encedana, Wolbachia-inducedmale killing occurs exclusively in fully developed embryoswith clearly visible tanned head capsules (Jiggins et al.2000). Wolbachia-induced male killing was also observedonly in larvae before hatching in the butterfly Hypolimnasbolina (Dyson et al. 2002). In all-female broods of the samespecies (H. bolina), however, Clarke et al. (1975) reportedthat males are killed mainly at the embryonic and first-instarlarval stages, although the presence of Wolbachia was notknown in this species at that time. Mitsuhashi et al. (2004)noted that in Wolbachia-infected lines of H. bolina, somemales died at the pupal stage when the Wolbachia densityin their mothers was decreased by tetracycline treatment.

Such differences in the timing of male killing may be de-

Table 3. Genetic sex ratios (WZ:ZZ) of larvae produced by 8 infected female O. furnacalis.

Proportion of eggs Genetic sex ratio (WZ:ZZ) of larvae

Line Brood Wolbachia infection Matured Hatched N Unhatched Day 0 Day 10 Day 20a

AK5014 1 Positive 1.00 0.84 110 7:11 21:29 n.e 21:0*** (1:2)2 Positive 0.97 0.92 89 2:3 30:20 12:0** 24:0*** (0:1)3 Positive 1.00 0.85 121 8:10 27:23 n.e 24:0*** (1:0)

AK5035 1 Positive 0.98 0.96 175 1:6 35:15 42:8*** 15:0**AK5062 1 Positive 1.00 0.77 128 3:10 26:24 30:7** n.e

2 Positive 0.98 0.78 108 4:19* 21:29 13:2* n.e3 Positive 1.00 0.89 116 1:12* 28:22 n.e 20:0*** (1b)4 Positive 0.99 0.94 94 0:5 27:23 n.e 15:0** (3b)

Total 0.99 0.87 941 26:76*** 215:185 91:17*** 95:0*** (2:3 + 4b)

Note: Genetic sex ratios were inferred by the presence (WZ, female) or absence (ZZ, male) of sex chromatin. Wolbachia infection in each broodwas checked by diagnostic PCR (30 samples/brood). N, number of eggs examined; n.e., not examined. Significant deviations from 1:1 are indicatedwith * (P < 0.05), ** (P < 0.01), and *** (P < 0.001), as determined by a �2 test.

aIn addition to ratios for normal-sized individuals, those for developmentally retarded individuals are shown separately in parentheses.bGenetic sex was not examined; all 4 dwarfs died before day 28.

Table 4. Genetic sex ratios (WZ:ZZ) of larvae produced by 6 cured female O. furnacalis.

Proportion of eggs Genetic sex ratio (WZ:ZZ) of larvae

Line Brood Wolbachia infection Matured Hatched N Unhatched Day 0 Day 10 Day 20a

AK5035 1 Negative 0.94 0.78 128 11:4 22:28 n.e. 0:26***2 Negative 1.00 0.93 113 7:0 33:17 0:31** n.e.3 Negative 1.00 0.91 138 11:0** 22:28 n.e. 0:45*** (0:3)

AK5062 1 Negative 1.00 0.91 97 7:2 27:23 2:12** n.e.2 Negative 1.00 0.97 141 3:2 18:32* 4:26*** 0:18*** (0:1)3 Negative 0.97 0.90 90 8:1* 22:28 0:14** n.e.

Total 0.98 0.90 707 47:9*** 144:156 6:83*** 0:89*** (0:4)

Note: Genetic sex ratios were inferred by the presence (WZ, female) or absence (ZZ, male) of sex chromatin. Wolbachia infection in eachbrood was checked by diagnostic PCR (30 samples/brood). N, number of eggs examined; n.e., not examined. Significant deviations from 1:1are indicated with * (P < 0.05), ** (P < 0.01), and *** (P < 0.001), as determined by a �2 test.

aIn addition to ratios for normal-sized individuals, those for developmentally retarded individuals are shown separately in parentheses.

Table 5. Sexual phenotypes of the offspring pro-duced by Wolbachia-infected O. furnacalis fe-males that were treated with tetracycline prior tooviposition.

No. of offspring

Day of oviposition(after treatment) Female Mosaic Male

1 65 0 02 106 0 03 56 20 274 26 9 41

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pendent on the density of Wolbachia. A density-dependenthypothesis for the timing of male killing was recently pro-posed for the Drosophila–Spiroplasma endosymbiotic sys-tem (Kageyama et al. 2007). In this system, timing of malekilling varies greatly from embryonic to adult stages de-pending on maternal age. Given that the Spiroplasma den-sity increases rapidly after adult emergence, the timing ofmale killing is likely to be dependent on symbiont densityin the Drosophila host (Kageyama et al. 2007). Accordingly,the variation in the timing of male killing found in Wolbachia-infected lines of O. furnacalis may be associated with thestochastic variation in Wolbachia density among males.

Mechanism of male killingThe occurrence of sexual mosaics that are genetically

male indicates that Wolbachia has feminizing abilities. Forthe genetic males, complete feminization must be lethal,whereas partial feminization induced by the antibiotic treat-ment can be managed. Given that none of the mosaics hadovaries (Kageyama and Traut 2004; Sakamoto 2006; thisstudy), feminization without lethal effects may be limited tospecific organs or tissues.

Male killing has been reported in a line of Ephestia kueh-niella artificially transfected with wSca, a Wolbachia strainthat has a feminizing effect on O. scapulalis (Fujii et al.2001; Kageyama et al. 2003). A small number of males thatbarely survived had no testes at the adult stage. This findingcan be explained well if we assume that these individualswere partially feminized genetic males and that the feminiz-ing effect of wSca underlies the occurrence of male killingin a foreign host (E. kuehniella).

In Drosophila melanogaster, many mutant strains thathave intersexual traits, such as transformer and doublesex,were revealed to be loss-of-function mutants of one of thesex-determining genes, which greatly contributed to ourunderstanding of the sex-determining system in Drosophila(reviewed by Steinmann-Zwicky et al. 1990; Schutt and No-thiger 2000). However, in Lepidoptera, even in the silk-worm, a model species for genetic studies, only a singleintersexual strain has been reported (Hirokawa 1995) despiteintensive surveys (e.g., Tazima 1964). Given that it is highlypossible that Wolbachia interferes with sex-determininggenes in Ostrinia spp., molecular analyses of gene expres-sion in Wolbachia-infected lines of Ostrinia spp. may con-tribute to our understanding of the sex-determining systemin lepidopteran insects in general.

Mechanism of female killingIn the progeny of Wolbachia-eliminated mothers, individ-

uals with female genotypes were killed during the larvalstage in both O. scapulalis (Kageyama and Traut 2004) andO. furnacalis (this study). Our data revealed that female kill-ing takes place over a wide range of larval stages. Two hy-potheses can account for the female killing. The‘‘compensation hypothesis’’ assumes there is a difference inthe host genotype between infected and uninfected matri-lines. More specifically, this hypothesis assumes that thereis a difference in either the W-linked or the cytoplasmicgene that influences the survival or sex determination of thedaughters, that the infected lines have lost the functions ofthe gene(s), and that Wolbachia compensates for the lost

functions. The second hypothesis, termed the ‘‘modificationhypothesis,’’ does not assume there are any differences inthe host genotype. This hypothesis assumes that Wolbachiasomehow modifies or imprints Ostrinia sp. oocytes suchthat any embryos (larvae) with a female genotype are killedduring development. When successfully transmitted to thedaughters, Wolbachia rescues the daughters. Otherwise, thedaughters die because of the modification. These hypothesesare mutually exclusive and are testable by transfection ex-periments.

The second hypothesis is reminiscent of the Medea(maternal-effect dominant embryonic arrest) gene, which isknown in beetles of the genus Tribolium (Beeman et al.1992). Selfish alleles at Medea loci are inherited in Men-delian fashion but ensure their own preferential propagationby causing maternal lethality to all hatchlings that do notinherit the selfish alleles (Beeman et al. 1992). The mech-anisms of maternal lethality and zygotic rescue in this ge-nus remain a mystery.

In the parasitic wasp Asobara tabida, the presence ofWolbachia is indispensable for host oogenesis (Dedeine etal. 2001, 2004). The ‘‘compensation hypothesis’’ and ‘‘mod-ification hypothesis’’ discussed above for explaining thefemale-killing phenomenon in Ostrinia spp. are also appli-cable to the obligate association between A. tabida andWolbachia (Charlat and Mercot 2001), but they remain un-tested. Recently, Pannebakker et al. (2007) showed that inA. tabita, Wolbachia manipulates apoptotic pathways duringoogenesis, making its presence essential for the wasp’s oo-cyte to mature. This finding does not rule out either of theabove 2 hypotheses but gives an intriguing suggestion re-garding the mechanism of female killing in the Ostrinia–Wolbachia system. Manipulation of apoptosis has alsobeen reported in the case of Spiroplasma-induced malekilling in Drosophila (Bentley et al. 2007) and thus mightunderlie the sex-specific death in Wolbachia-infectedstrains of Ostrinia spp.

Killing of females that failed to inherit Wolbachia wouldbe evolutionarily advantageous because it would increasethe relative frequency of infected females. The type of malekilling found in Ostrinia spp. can be regarded as advancedand, to date, has not been reported in other arthropod–Wolbachia systems.

The possibility of an integrative explanation of sex-specificlethality

Similarities in the characteristics and timing of male kill-ing and female killing may imply that these sex-specific le-thal traits represent mirror images of a single biologicalphenomenon. In this context, the ‘‘compensation hypothe-sis’’ seems likely for the mechanism of female killing. Onepossibility is directly relevant to sex-determination proc-esses. As we have shown, Wolbachia has a feminizing effecton genetic males. In the infected lines, genetic females mayhave lost a function for female sex determination, which iscompensated for by Wolbachia. Thus, in the cured lines, ge-netic females may develop as males, and the resulting incon-gruence between genetic sex and phenotypic sex would leadto female-specific death. Alternatively, Wolbachia maycompensate for a defect of dosage compensation. In Droso-phila, X-linked genes in XY males are hypertranscribed to

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equalize the gene expression in the 2 sexes (reviewed byBaker et al. 1994; Marin et al. 2000). Wolbachia might killmales or females by manipulating the dosage compensationsystem in Ostrinia spp. In the silkworm Bombyx mori, how-ever, not all Z-linked genes appear to be dosage compen-sated (Suzuki et al. 1998, 1999), and thus the necessity fordosage compensation may not be absolute in lepidopteraninsects.

AcknowledgementsWe thank Ryo Nakano and Laurent Pelozuelo for their

kind gift of O. furnacalis larvae, and Prof. Sadahiro Tatsukifor helpful advice. This study was supported in part byGrants-in-Aid for Scientific Research (Nos. 15780038 and16208005) from the Ministry of Education, Culture, Sports,Science, and Technology of Japan.

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